The mosquito-borne flaviviruses include the NIAID list of Class A, B, or C emerging human pathogens such as Yellow fever virus (YFV), West Nile virus (WNV) and Dengue (DEN1-4) viruses that cause serious illnesses with considerable morbidity and mortality. These viruses, in addition to flu-like symptoms, can cause hemorrhagic fevers (YFV and DEN) or encephalitis and/or meningo-encephalitis in susceptible humans. One of the long term goals of this laboratory has been to develop antiviral therapeutics through understanding of key pathways in the viral life cycle using in vitro enzymatic assays that mimic those utilized by these viruses in their natural hosts. Two of the nonstructural proteins, NS3 and NS5, have multiple enzyme activities that are required for the virus life cycle. NS3, in association with NS2B, is a serine protease that is required for polyprotein processing, a key early event in the virus life cycle. Moreover, NS3 exhibits an RNA-stimulated NTPase, RNA helicase and the 5'-RNA triphosphatase (5'-RTPase) activities. The NTPase and 5'-RTPase activities are stimulated by interaction with NS5, the viral 5'-RNA methyltransferase and the RNA dependent RNA polymerase (RdRP). These enzyme activities are excellent targets for development of antiviral therapeutics. In Specific Aim 1, we propose test the hypothesis that small molecule compounds could potentially inhibit the interaction between the NS2B cofactor and the NS3 protease (NS3-pro) domain or by mimicking the substrate-protease interactions or both. To this hypothesis, we will screen approximately 175,000 compounds in the National Screening Laboratory using the in vitro protease assay. By using chemoinformatics and computer-assisted docking studies and the crystal structure of the DEN protease domain, we will select about 100 compounds with greater than or equal to 50% inhibition of (i) interaction between the NS2B cofactor-NS3-pro domain and (ii) interaction between the substrate-protease active site interaction by measuring the Kcat and Km values in the presence and absence of inhibitors. Further testing of selected compounds will be performed using cytotoxicity, viral infectivity and replicon-based assays. In Specific Aim 2, the hypothesis that small molecule compounds that could potentially inhibit NS3/NS5 interaction and block their individual functions will be tested. We will identify approximately 200 compounds using a similar strategy as in Aim 1 but by using the in vitro assay based on measurement of the Pi released from the hydrolysis of either NTP or the 5'-triphosphorylated RNA and the recently reported crystal structures of YFV and DEN2 RNA helicases and those of hepatitis C virus and bovine viral diarrhea virus (BVDV) RdRP for docking studies. Inhibitors of interaction between NS3-RNA, NS3-NTP, NS3-NS5 will be analyzed by immunoprecipitation, surface plasmon resonance and gel shift assays. Further testing of selected compounds will be performed using cytotoxicity, viral infectivity and replicon-based reporter assays. This proposed research is likely lead to identification of novel inhibitors that will be useful for co-crystallization and structure-function studies that could provide valuable insight into the nature of molecular interactions among flavivirus nonstructural proteins in the flavivirus life cycle.